Biodegradable superparamagnetic metal oxides as contrast agents for MR imaging

ABSTRACT

This invention relates to materials exhibiting certain magnetic and biological properties which make them uniquely suitable for use as magnetic resonance imaging (MRI) agents to enhance MR images of animal organs and tissues. More particularly, the invention relates to the in vivo use of biologically degradable and metabolizable superparamagnetic metal oxides as MR contrast agents. Depending on their preparation, these metal oxides are in the form of superparamagnetic particle dispersoids or superparamagnetic fluids where the suspending medium is a physiologically-acceptable carrier, and may be uncoated or surrounded by a polymeric coating to which biological molecules can be attached. These materials are administered to animals, including humans, by a variety of routes and the metal oxides therein collect in specific target organs to be imaged; in the case of coated particles, the biological molecules can be chosen to target specific organs or tissues. The biodistribution of the metal oxides in target organs or tissues results in a more detailed image of such organs or tissues because the metal oxides, due to their superparamagnetic properties, exert profound effects on the hydrogen nuclei responsible for the MR image. In addition, the dispersoids and fluids are quite stable and, in the case of the fluids, can even be subjected to autoclaving without impairing their utility. Furthermore, the materials are biodegradable and, in the case of iron oxide compounds, can eventually be incorporated into the subject&#39;s hemoglobin, making them useful in treating anemia. Thus, the materials are well-suited for in vivo use.

CROSS REFERENCE TO RELATED APPLICATION

This application is a continuation of applicants' prior co-pending U.S.application Ser. No. 067,586 filed June 26, 1987, now U.S. Pat. No.4,827,945, which in turn, is a continuation-in-part of the applicants'prior co-pending U.S. application Ser. No. 882,044 filed July 3, 1986,now U.S. Pat. No. 4,770,183, the disclosures of which are incorporatedherein by reference.

INTRODUCTION

This invention relates to materials exhibiting certain magnetic andbiological properties which make them uniquely suitable for use asmagnetic resonance imaging (MRI) agents to enhance MR images of animalorgans and tissues. More particularly, the invention relates to the invivo use of biologically degradable and metabolizable superparamagneticmetal oxides as MR contrast agents. Depending on their preparation,these metal oxides are in the form of superparamagnetic particledispersoids or superparamagnetic fluids where the suspending medium is aphysiologically-acceptable carrier. These dispersoids and fluids areadministered to animals, including humans, by a variety of routes andthe metal oxides therein collect in specific target organs to be imaged.The biodistribution of the metal oxides in target organs or tissuesresults in a more detailed image of such organs or tissues because themetal oxides due to their superparamagnetic properties, exert profoundeffects on the hydrogen nuclei responsible for the MR image. Inaddition, the dispersoids and fluids are quite stable and, in the caseof the fluids, can even be subjected to autoclaving without impairingtheir utility. Thus, the materials are well-suited for in vivo use.

The combination of superparamagnetism and biodegradability makes thematerials described herein particularly advantageous for use as MRcontrast agents. Superparamagnetism, which results in profoundcapabilities to alter MR images, makes it possible to use thesematerials in concentrations lower than those required for MRI with othertypes of magnetic materials. Biodegradability results in optimumretention times within the organs and tissues to be imaged, i.e., thematerials remain within the organ or tissue sufficiently long to permitan image to be obtained, yet are eventually cleared from or metabolizedwithin the organ or tissue. Remarkably, when iron-based agents areadministered, the iron thereon is eventually metabolized andincorporated into the subject's hemoglobin.

These materials can, therefore, be used for a variety of clinicaldiagnostic purposes including, but not limited to, detection ofcancerous lesions in liver and other reticuloendothelial tissue,detection of cancerous or other lesions in the intestine, detection ofliver diseases, such as cirrhosis and hepatitis, and assessment of liverregeneration. Those that are iron-based are also clinically useful asantianemia agents.

2 BACKGROUND OF THE INVENTION 2.1 In Vivo NMR Imaging: GeneralConsiderations

Nuclear magnetic resonance (NMR) is now widely used for obtainingspatial images of human subjects for clinical diagnosis. Clinical usageof NMR imaging, also called magnetic resonance imaging or, simply, MRI,for diagnostic purposes has been reviewed [see e.g., Pykett, et al.,Nuclear Magnetic Resonance, pgs. 157-167 (April, 1982) and T. F.Budinger, et al., Science, pgs. 288-298, (October, 1984)]. Severaladvantages of using such a procedure over currently used diagnosticmethods, e.g., x-ray computer-aided tomography (CT), are generallyrecognized. For instance, the magnetic fields utilized in a clinical NMRscan are not considered to possess any deleterious effects to humanhealth (see Budinger, supra., at 296). Additionally, while x-ray CTimages are formed from the observation of a single parameter, x-rayattenuation, MR images are a composite of the effects of a number ofparameters which are analyzed and combined by computer. Choice of theappropriate instrument parameters such as radio frequency (Rf), pulsingand timing can be utilized to enhance (or, conversely, attenuate) thesignals of any of the image-producing parameters thereby improving theimage quality and providing better anatomical and functionalinformation. Finally, the use of such imaging has, in some cases, provento be a valuable diagnostic tool as normal and diseased tissue, byvirtue of their possessing different parameter values, can bedifferentiated in the image.

In MRI, the image of an organ or tissue is obtained by placing a subjectin a strong external magnetic field and observing the effect of thisfield on the magnetic properties of the protons (hydrogen nuclei)contained in and surrounding the organ or tissue. The proton relaxationtimes, termed T₁ and T₂, are of primary importance. T₁ (also called thespin-lattice or longitudinal relaxation time) and T₂ (also called thespin-spin or transverse relaxation time) depend on the chemical andphysical environment of organ or tissue protons and are measured usingthe Rf pulsing technique; this information is analyzed as a function ofdistance by computer which then uses it to generate an image.

The image produced, however, often lacks definition and clarity due tothe similarity of the signal from other tissues. To generate an imagewith good definition, T₁ and/or T₂ of the tissue to be imaged must bedistinct from that of the background tissue. In some cases, themagnitude of these differences is small, limiting diagnosticeffectiveness. Thus, there exists a real need for methods which increaseor magnify these differences. One approach is the use of contrastagents.

2.2. MRI Contrast Agents

As any material suitable for use as a contrast agent must affect themagnetic properties of the surrounding tissue, MRI contrast agents canbe categorized by their magnetic properties.

Paramagnetic materials have been used as MRI contrast agents because oftheir long recognized ability to decrease T₁ [Weinmann et al., Am. J.Rad. 142, 619 (1984), Greif et al. Radiology 157, 461 (1985), Runge, etal. Radiology 147, 789 (1983), Brasch, Radiology 147, 781 (1983)].Paramagnetic materials are characterized by a weak, positive magneticsusceptibility and by their inability to remain magnetic in the absenceof an applied magnetic filed.

Paramagnetic MRI contrast agents are usually transition metal ions ofiron, manganese or gadolinium. They may be bound with chelators toreduce the toxicity of the metal ion (see Weinman reference above).Paramagnetic materials for use as MRI contrast agents are the subject ofa number of patents and patent applications. (See EPA No. 0 160 552; UKApplication No. 2 137 612A; EPA No. 0 184 899; EPA No. 0 186 947; U.S.Pat. No. 4,615,879; PCT No. WO 85/05554; and EPA No. 0 210 043).

Ferromagnetic materials have also been used as contrast agents becauseof their ability to decrease T₂ Medonca-Dias and Lauterbur, Magn. Res.Med. 3, 328, (1986); Olsson et al., Mag Res. Imaging 4, 437 (1986);Renshaw et al. Mag Res. Imaging 4, 351 (1986) and 3, 217 (1986)].Ferromagnetic materials have high, positive magnetic susceptibilitiesand maintain their magnetism in the absence of an applied field.Ferromagnetic materials for use as MRI contrast agents are the subjectof recent patent applications [PCT No. WO 86/01112; PCT No. WO85/043301].

A third class of magnetic materials termed superparamagnetic materialshave been used as contrast agents [Saini et al Radiology, 167, 211(1987); Hahn et al., Soc. Mag Res. Med. 4(22) 1537 (1986)]. Likeparamagnetic materials, superparamagnetic materials are characterized byan inability to remain magnetic in the absence of an applied magneticfield. Superparamagnetic materials can have magnetic susceptibilitiesnearly as high as ferromagnetic materials and far higher thanparamagnetic materials [Bean and Livingston J. Appl. Phys. suppl to vol.30, 1205, (1959)].

Ferromagnetism and superparamagnetism are properties of lattices ratherthan ions or gases. Iron oxides such as magnetite and gamma ferric oxideexhibit ferromagnetism or superparamagnetism depending on the size ofthe crystals comprising the material, with larger crystals beingferromagnetic [G. Bate in Ferromagnetic Materials. vol. 2, Wohlfarth(ed.) p.439].

As generally used, superparamagnetic and ferromagnetic materials alterthe MR image by decreasing T₂ resulting in image darkening. Wheninjected, crystals of these magnetic materials accumulate in thetargeted organs or tissues and darken the organs or tissues where theyhave accumulated. Abnormal volumes of liver, such as tumors, aredeficient in their ability to take up the magnetic materials and appearlighter against normal background tissue than they would withoutcontrast agent.

2.3 Superparamagnetic Materials

As stated supra, superparamagnetic materials possess somecharacteristics of paramagnetic and some characteristics offerromagnetic materials. Like paramagnetic materials, superparamagneticmaterials rapidly lose their magnetic properties in the absence of anapplied magnetic field; they also possess the high magneticsusceptibility and crystalline structure found in ferromagneticmaterials. Iron oxides such as magnetite or gamma ferric oxide exhibitsuperparamagnetism when the crystal diameter falls significantly belowthat of purely ferromagnetic materials.

For cubic magnetite (Fe₃ O₄) this cut-off is a crystal diameter of about300 angstroms [Dunlop, J. Geophys. Rev. 78 1780 (1972)]. A similarcut-off applies for gamma ferric oxide [Bare in Ferromagnetic Materials,vol. 2, Wohfarth (ed.) (1980) p. 439]. Since iron oxide crystals aregenerally not of a single uniform size, the average size of purelyferromagnetic iron oxides is substantially larger than the cut-off of300 angstroms (0.03 microns). For example, when gamma ferric oxide isused as a ferromagnetic material in magnetic recording, (e.g., PfizerCorp. Pf 2228), particles v 30 are needle-like and about 0.35 micronslong and 0.06 microns thick. Other ferromagnetic particles for datarecording are between 0.1 and 10 microns in length [Jorgensen, TheComplete Handbook of Magnetic Recording, p. 35 (1980)]. For a given typeof crystal, preparations of purely ferromagnetic particles have averagedimensions many times larger than preparations of superparamagneticparticles.

The theoretical basis of superparamagnetism has been described in detailby Bean and Livington [J. Applied Physics, Supplement to volume 30, 1205(1959)]. Fundamental to the theory of superparamagnetic materials is thedestabilizing effect of temperature on their magnetism. Thermal energyprevents the alignment of the magnetic moments present insuperparamagnetic materials. After the removal of an applied magneticfield, the magnetic moments of superparamagnetic materials still exist,but are in rapid motion, causing a randomly oriented or disorderedmagnetic moment and, thus, no net magnetic field. At the temperatures ofbiological systems and in the applied magnetic fields of MR imagers,superparamagnetic materials are less magnetic than their ferromagneticcounterparts. For example, Berkowitz et al. [(J. App. Phys. 39, 1261(1968)] have noted decreased magnetism of small superparamagnetic ironoxides at elevated temperatures. This may in part explain why workers inthe field of MR imaging have looked to ferromagnetic materials ascontrast agents on the theory that the more magnetic a material is pergram, the more effective that material should be in depressing T₂[Drain, Proc. Phys. Soc. 80, 1380 (1962); Medonca-Dias and Lauterur,Mag. Res. Med. 3, 328 (1986)].

2.4. Water-Based Superparamagnetic Solutions

It has been recognized for some time that superparamagnetic particlescan be fashioned into magnetic fluids termed ferrofluids [see Kaiser andMiskolczy, J. Appl. Phys. 41 3 1064 (1970)]. A ferrofluid is a solutionof Very fine magnetic particles kept from settling by Brownian motion.To prevent particle agglomeration through Van der Waals attractiveforces, the particles are coated in some fashion. When a magnetic fieldis applied, the magnetic force is transmitted to the entire volume ofliquid and the ferrofluid responds as a fluid, i.e. the magneticparticles do not separate from solvent.

Another approach to synthesizing water-based magnetic compounds isdisclosed by Gable et al (U.S. Pat. No. 4,001,288). Here, the patentdiscloses that magnetite can be reacted with a hydroxycarboxylic acid toform a water soluble complex that exhibits ferromagnetic behavior bothin the solid form and in solution.

2.4.1. Problems Manipulating Aqueous Solutions of SuperparamagneticMaterials

Approaches to the synthesis of aqueous fluids of superparamagnetic ironoxides often involve surrounding iron oxide crystals with polymer orsurfactants in an effort to block the attractive forces between thecrystals that promote aggregation. In many cases however, the polymerdoes not completely coat the oxide and the resultant material maintainsmuch of the sensitivity to clumping or aggregation characteristic of theuncoated iron oxide. The tendency to clump, and other peculiarproperties of iron oxide solutions, hamper the manipulations of thesesolutions needed in pharmaceutical manufacture.

The manufacture of a magnetic pharmaceutical solution such as an MRIcontrast agent requires an extremely stable solution so certainmanipulations, common in pharmaceutical manufacture, can be carried out.Solution stability is defined as the retention of the size of themagnetic material in solution; in an unstable solution the material willclump or aggregate. Such changes in the size of magnetic material alterits biodistribution after injection, an intolerable situation for an MRIcontrast agent. A high degree of stability is required to perform commonoperations associated with pharmaceutical manufacture such as dialysis,concentration, filtration, centrifugation, storage of concentrates priorto bottling, and long term storage after bottling. Particular problemsare posed by the need to sterilize aqueous solutions of metal oxide,e.g. iron oxide, for pharmaceutical use.

Additionally, concentrated solutions of aqueous superparamagneticmaterials cannot be sterilized by filtration even when the solution iscomprised of materials smaller than the pore of the filter. Thisphenomena is related to the concentration of the solution, for dilutesolutions can be filter sterilized. Filter-sterilized, dilute materialcan be reconcentrated and dispensed into sterile bottles, but suchoperations offer many chances to recontaminate the product. Autoclavingsolutions of superparamagnetic materials after bottling is preferable,since sterilization is achieved after final bottling, and there islittle opportunity for contamination of the final product. Autoclavinginvolves heating sealed solutions to 121° C. for 30 minutes Such extremetemperatures induce aggregation or clumping of the superparamagneticoxides, making them unusable as an injectable material.

2.5. Paramagnetic Ferric Oxides

Paramagnetic iron oxides or ferric oxides are currently used in thetreatment of anemia under many trade names such as Imferon. Whendissolved in aqueous solution, such materials can be represented asFeO:OH and are termed ferric oxyhydroxides. They are paramagnetic andexert small, if any, effects of proton relaxivity. They are stable,undergo the manipulations discussed supra for pharmaceuticalmanufacture, and are commercially available as drugs used in thetreatment of anemia.

3. NOMENCLATURE

The term "biodegradable" in reference to the materials of this inventionis defined as being metabolized and/or excreted by the subject within 30days or less; for superparamagnetic iron oxides, the term is furtherdefined as being incorporated into the hemoglobin of a subject within 30days or less after administration.

The term "blocking agent" is defined as any material which whenadministered parenternally to a subject, will competitively bind to thereceptors of the cells of the reticuloendothelial system which recognizeand bind MRI contrast agents.

The term "superparamagnetic fluid" defines any metal oxide fluidproduced by the methods described in section 6.3 herein, which has thecharacteristics described in section 6.4 herein.

4. Summary of the Invention

It is an object of this invention to provide an in vivo MR imagingtechnique for diagnostic purposes which will produce a clear,well-defined image of a target organ or tissue. Specifically, it is anobject of this invention to provide an imaging method using MR contrastagents which are easily administered, exert a significant effect on theimage produced and which distribute in vivo to specific organs ortissues. These contrast agents are stable in vivo, can be easilyprocessed for in vivo use, and overcome problems of toxicity andexcessively long retention in the subject (i.e. are biodegradable). Itis further an object of this invention to provide a means whereby thesecontrasts agents can be directed, in vivo, to a specific target organ ortissue.

This invention provides a novel MR imaging method using biodegradablesuperparamagnetic metal oxides as contrast agents which fulfill theforegoing objectives. Such materials, it has been discovered, combine anoptimal balance of features and are particularly well-suited for use asMR contrast agents. Remarkably, it has been found that thesesuperparamagnetic materials exert a greater effect on T₂ thanferromagnetic or paramagnetic materials, thereby producing awell-resolved, negative contrast image of an in vivo target organ ortissue. It has also been surprisingly found that the materials used inthe methods of this invention exhibit highly desirable in vivo retentiontimes, i.e., they remain intact for a sufficient time to permit theimage to be taken, yet are ultimately biodegradable. Remarkably, oncedegraded, iron-based materials serve as a source of nutritional iron.Additionally, they are sufficiently small to permit free circulationthrough the subject's vascular system and rapid absorption by theorgan/tissue being imaged, allowing for maximum latitude in the choiceof administration routes and ultimate targets.

In one embodiment, the materials used as MR imaging agents comprisesuperparamagnetic metal oxide particles which comprise superparamagneticcrystalline cores. Each core is composed of magnetically active metaloxide crystals which range from about 10 to about 500 angstroms indiameter. The cores may be uncoated or, alternatively, coated orassociated with a polysaccharide, a protein, a polypeptide or anycomposite thereof. By way of illustration, the polysaccharide coatingmay comprise dextran of varying molecular weights and the proteincoating may comprise bovine or human serum albumin. With coatings, theoverall particle diameter ranges from about 10 upward to about 5,000angstroms. In the case of coated particles, the coatings can serve as abase to which various biological molecules can be attached. Thebiological molecules can be used to direct the particles to the desiredtarget and are preferentially recognized and bound by the target organor tissue. Such molecules include proteins, polysaccharrides, hormones,antibodies, etc.

Preferred superparamagnetic particles comprise iron oxides with crystalsizes ranging from about 50 to about 500 angstroms. These iron oxideparticles have surface areas greater than 75 m² /gram. In aqueoussolution, these iron oxide particles have a size range between about 50and about 5,000 angstroms, including coatings, if any. Thesuperparamagnetic iron oxides have magnetic saturations between about 5and about 90 electromagnetic units (EMU) per gram of oxide at roomtemperature (approximately 25° C.) and possess a magnetic squareness ofless than 0.10, i.e., lose greater than 90% of their magnetism when anapplied magnetic field is removed.

Superparamagnetic particles with these general dimensions overcomeproblems associated with the use of ferromagnetic and paramagneticmaterials as MR contrast agents. Specifically, superparamagneticparticles, because they are smaller than ferromagnetic particles, aremore able to avoid uptake by the subject's reticuloendothelial cells andmay be more effectively targeted to other organ and tissue sites withinthe body. Also, because the superparamagnetic particles are smaller thanferromagnetic particles, they have higher surface area per unit mass andare more easily and rapidly digested by chemical or metabolic processes.However, the superparamagnetic particles used herein, because they arelarger than paramagnetic ions, are not so rapidly metabolized in thetarget organ or tissue as to prevent convenient imaging.

Uncoated or coated particles may be suspended in an appropriate medium(e.g., saline) to form a particle dispersoid that has the properties ofa solution. The particles do not settle upon standing and do not scattervisible light (i.e., the solution appears translucent). Solvent can beadded (decreasing) or removed (increasing) particle concentration.

This dispersoid of particles may be administered to the subject beingstudied. Depending on the route of administration, the particles aredistributed to various target organs, where absorption occurs. Forexample, when the superparamagnetic particles are administeredintravascularly (e.g., intravenously or intra-arterially), they areselectively distributed to reticuloendothelial organs, including liver,spleen, lymph nodes and bone marrow and, to a lesser extent, lung.However, when the superparamagnetic particles are administered via thegastrointestinal tract, e.g., orally, by intubation or by enema, theycan be used as imaging agents for the organs and tissues of thegastrointestinal tract.

The use of sub-micron sized particles is particularly important when theroute of administration is intravascular, as such particles can freelycirculate in the subject's vascular system, since they are small enoughto pass through the capillary network. Thus, such contrast agents can becarried to targeted organs or tissue after being intravascularlyadministered with a minimum of trouble or delay.

In one embodiment, a dextran-coated iron oxide particle dispersoid isinjected into a subject's bloodstream and the particles localize in theliver. The particles are absorbed by the reticuloendothelial cells ofthe liver by phagocytic uptake; a particular benefit of this mode ofuptake is that phagocytized iron is metabolized and cleared from theliver much more slowly (but not so slowly as to lead to undesirably longretention times) than prior art paramagnetic ions. Additionally, thedextran-coated particles can be preferentially absorbed by healthycells, with less uptake into cancerous (tumor) cells. This preferentialuptake enhances the contrast between healthy and cancerous tissue andallows for better definition of the tumor location on the image.

In another embodiment of this invention, the materials comprise stable,biodegradable superparamagnetic metal oxides, preferably ferric oxides,in the form of superparamagnetic fluids. These superparamagnetic fluidsexhibit some of the magnetic properties of superparamagnetic ferrofluids(e.g., the metal oxides in them cannot be removed from solution bymagnetic manipulation), yet the metal oxides in them can be easilyreclaimed from the bulk fluid by physical means (e.g. centrifugation)and, ultimately redispersed in the bulk fluid. When dispersed, the metaloxides will not scatter visible light, indicating the individual metaloxide "particles" are quite small (generally between 50 and 4000angstroms in diameter.)

The metal oxides of the invention exist in the bulk fluid as ioniccrystals, having both ionic and crystalline characteristics. In commonwith magnetite (Fe₃ O₄) and gamma ferric oxide (gamma Fe₂ O₃) they havehigh magnetic susceptibility. In common with ionic forms of ferricoxide, the so-called ferric oxyhydroxides, they cause retention ofanions. The counterion to the crystals can be any one of a number oforganic anions.

In a preferred embodiment, the metal oxide is a superparamagnetic ferricoxyhydroxide and the counterion is citrate. The use of citratecounterions also confers a distinct advantage to the fluids as itrenders them highly stable. In fact, the citrated fluids can withstandautoclaving greatly facilitating sterile administration.

The metal oxides in the superparamagnetic fluids may also be surroundedby a coating comprising a polysaccharide, a protein, a polypeptide, anorganosilane, or any composite thereof. These polymeric coatings serve adual purpose, helping to stabilize the superparamagnetic fluids as wellas serving as a base to which biological molecules can be attached.These biological molecules can be used to direct particles to thedesired target and are preferentially recognized and bound by the targetorgan or tissue. These molecules include proteins, polysaccharides,hormones, antibodies, etc.

The superparamagnetic fluids, whether comprised of coated or uncoatedmetal oxides, can be administered to a subject by any of the meansdescribed supra for the metal oxide dispersoids. Furthermore, in generalthe fluids are quite stable and can be prepared well in advance of useand stored.

The superparamagnetic fluids, containing both coated and uncoated metaloxides, are produced by a unique three step process from a mixture ofFe²⁺ and Fe³⁺ salts. In addition, this process permits incorporation ofother metals similar to iron (such as cobalt (Co) and manganese (Mn)into the fluids by replacing some of the divalent iron salts withdivalent salts of these metals. In the process, the salts areprecipitated in base to form the corresponding oxides. These oxides arethen dispersed and oxidized by sonication of the mixture; the result,remarkably, is a superparamagnetic ferric oxyhydroxide. Insoluble oxidescan then be removed by centrifugation and the final fluid is dialyzedagainst a neutral or alkaline buffer suitable for in vivo use.

In a preferred embodiment, the salt mixture is FeCl₂ /FeCl₃ in a 1:2ratio and the buffer is 10 mM ammonium citrate at pH 8.2. The result isa superparamagnetic fluid of unusual stability, characterized by itscapacity to withstand autoclaving.

Once administered both the metal oxides of the superparamagneticparticle dispersoids and the superparamagnetic fluids collect in thetarget organ or tissue and exert a profound contrast effect to permit animage to be taken. The superparamagnetic metal oxides act primarily toenhance T₂ relaxation, but T₁ is also affected (although to a lesserextent).

Another embodiment of this invention presents a method for extending thelifetime of the superparamagnetic metal oxides in the subject's serum.The method comprises administering a dose of paramagnetic metal oxide inthe same form as the superparamagnetic imaging agent (i.e. appropriateparticle size and, if applicable, the same coating) as a blocking agentprior to the administration of the imaging agent. This blocking agentwill compete with the imaging agent for binding to thereticuloendothelial system (RES) receptors. Since the RES is responsiblefor removing impurities from the blood, the binding of the blockingagent greatly increases the serum lifetime of the imaging agent.Potential applications of this procedure include, but are not limitedto, use of MRI to diagnose blood circulation disorders and strokes.

5. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a graphical representation comparing the effect offerromagnetic and superparamagnetic contrast agents on T₂ ;

FIG. 2 is a composite of five in vivo MR images of a cancerous rat liverobtained on a Technicare MR Imager;

FIGS. 2A and 2B were obtained without the use of contrast agents andwere taken at different settings of instrument;

FIGS. 2C and 2D were obtained after the intravenous administration ofthe dextran-coated particle produced in Example 6.1. at a dosage of 0.5mg/kg; the tumor can clearly be seen;

FIG. 2E is the image reproduced in FIG. 2C showing the tumor highlightedby crosshairs;

FIG. 3 is a graphical representation of the % T₂ reduction in liver andspleen tissue for three different dosages of an uncoatedsuperparamagnetic particle as a function of time after administration;

FIG. 4 presents hysteresis curves for paramagnetic, ferromagnetic andsuperparamagnetic iron oxides;

FIGS. 5A and 5B shows the effect of autoclaving on superparamagneticfluids prepared as in Example 7.10 having varying concentrations ofcitrate;

FIG. 6 presents a schematic diagram of the apparatus used in Example7.10; and

FIG. 7 is a graphical representation of the T₂ of rat blood as afunction of time after the injection of a dextran-coatedsuperparamagnetic iron oxide particle with and without the use of ablocking agent.

6. DETAILED DESCRIPTION OF THE INVENTION 6.1. Preparation of CoatedSuperparamagnetic Iron Oxide Particles

The synthesis of superparamagnetic iron oxide particles for use as MRIcontrast agents is accomplished by mixing ferrous and ferric salts withbase to form a black, magnetic oxide of iron. Crystals result from suchprecipitations, for when the material is subjected to X-ray diffractionanalyses long range order is apparent. A diameter of between about 50and about 300 angstroms for such crystals has been calculated althoughcrystals may range in diameter from about 10 to about 500 angstroms. Theiron oxides have correspondingly high surface areas, greater than about75 m² /gm.

The presence of ferrous salts prior to base addition insures theformation of a black, crystalline magnetic iron oxide. Without theferrous ion, paramagnetic ferric oxide gels (noncrystalline materials)result (as described e.g., in U.S. Pat. No. 2,885,393). The presence ofdivalent iron, so essential to the formation of the superparamagneticmaterial, can then be removed by exposure of the material to oxidizingconditions. Oxidation of the iron to produce a ferric oxide afterformation of the crystal does not alter the usefulness of the materialas a contrast agent in MRI or the superparamagnetism.

It is to be understood throughout this detailed description, that theuse of superparamagnetic iron oxides as MR contrast agents is but oneembodiment of the invention and that superparamagnetic oxides of othermagnetic metals, e.g., cobalt or gadolinium, may be substituted for ironoxides.

There are two general strategies for the formation of the coatedsuperparamagnetic iron oxide particles suitable for MRI.

1. Synthesis of iron oxide by precipitation in the presence of polymerslike dextran, or polyglutaraldehyde or other material. Such synthesesinclude those described by London et al., U.S. Pat. No. 2,870,740,Molday, U.S. Pat. No. 4,452,773, Cox et al., Nature, 208, 237 (1965) andRembaum, U.S. Pat. No. 4,267,234; all of which are incorporated hereinby reference.

2. Synthesis of the iron oxide by precipitation followed by coating witha polymer like dextran or other material. This type of synthetic routeis utilized by Elmore, Phys. Rev. 54, 309 (1938) and Ohgushi et al., J.Mag Res., 9, 599 (1978); both of which are incorporated herein byreference.

With proteins and dextrans, synthesis of the oxide in the presence ofthe polymer seems to effect a tight association between the polymer andthe oxide. The synthesis of oxide first, followed by exposure to aprotein or dextran yields a coated particle with the coating being heldto the particle surface by relatively weak adsorption phenomena.However, if the oxide and adsorbed polymer can be manipulated, storedand injected in the presence of nonadsorbed polymer, the weakness of theassociation between oxide and polymer is not a problem. For example,when the particles of Section 6.1.3. (uncoated) are diluted 1:1 into aneutral buffer containing 1% w/v human serum albumin (HSA), considerableprotein will adsorb to the oxide surface. This approach to the synthesisof an albumin coated magnetic particle is a practical one for an imagingagent. The HSA coated particle (plus HSA in solution) can be injectedinto a patient and the HSA in solution mixes with HSA in serum. Whenparticles are made by this approach the loosely associated HSA can beremoved by treatments such as moderate temperature (50° C.) or high salt(1 M NaCl).

The coating methods are general and can be performed with a variety ofphysiologically acceptable proteins and carbohydrates, particularlythose with molecular weights from about 5,000 to about 250,000 daltons.Other polymeric coatings include, but are not limited to,albumin/dextran composites, ficoll, dextrin, starch, glycogen andpolyethylene glycol.

6.1.1. Preparation of Polysaccharide-Coated Particles

Polysaccharide-coated superparamagnetic iron oxide particles (about 10to about 5000 angstroms in diameter) useful as MR contrast agents areprepared by a single-step process according to the procedure of Molday[U.S. Pat. No. 4,452,773] incorporated herein by reference above. In apreferred embodiment, dextranized divalent (Fe²⁺) and trivalent (Fe³⁺)iron salts, e.g. FeCl₂ and FeCl₃, are precipitated from an aqueoussolution containing a mixture of the iron salts and dextran (molecularweight of dextran can vary from 5,000 to 250,000 daltons) by thedropwise addition (to pH=10) of base ammonium hydroxide at 60°-65° C.,followed by centrifugation at 1500×g for 15 minutes to remove theoversized particles which are subsequently discarded. The remainingparticles are dialyzed against distilled water and can be concentratedby ultrafiltration. Any unbound dextran can be removed by gel filtrationchromatography in a chloride/acetate buffer.

The ratio of Fe³⁺ to Fe²⁺ is preferentially maintained at about 2:1, butcan be varied from about 0.5:1 to about 4.0:1 without substantialchanges in product quality and efficiency as contrast agents.

Likewise, bases other than ammonium hydroxide (NH₄ OH) can be used, butNH₄ OH is preferred because the ammonium ion has a slight dispersingeffect on iron oxides which increases the yield.

As mentioned above, various magnetically active metals notably Co, andMn, may be substituted for Fe without any deleterious effect on theefficiency of the particles as contrast agents. Use of otherpolysaccharides such as starches, glycogen or dextrins is alsocontemplated.

6.1.2. Preparation of Protein-Coated Particles

Protein-coated superparamagnetic iron oxide particles are prepared by asingle-step process similar to that of Molday [U.S. Pat. No. 4,452,733].The protein-coated particles can be prepared like the dextran coatedwherein the iron salts (e.g., FeCl₂ and FeCl₃) and the protein aredissolved in water and the coated iron oxide particles are precipitatedby the dropwise addition of base (NH₄ OH) to pH=10. In an alternativeembodiment the protein can be dissolved in the base and an aqueoussolution of the iron salts can be added dropwise to form a coatedparticle.

In either method, the oversized particles are subsequently collected bycentrifugation at 1500×g and the remaining particles are subjected todialysis against distilled water followed by ultrafiltration. Anyunbound protein can be removed by gel filtration chromatography in achloride/acetate buffer.

As with the polysaccharide coated particles, both the coatingcomposition and the Fe³⁺ /Fe²⁺ ratio (about 2/1) can be varied fromabout 0.5:1 to about 4:1 without any deleterious effect on theefficiency of these particles as contrast agents.

As mentioned above, various magnetically active metals notably Co, andMn, may be substituted for Fe without any deleterious effect on theefficiency of the particles as contrast agents.

6.1. 3. Preparation of Uncoated Particles

Uncoated superparamagnetic iron oxide particles are prepared by mixingan solution of ferric chloride (FeCl₃) with ferrous chloride (FeCl₂) inHCl and precipitating in 0.7 molar aqueous ammonia. The baseprecipitation offers a dual advantage in that the base reacts with theiron chlorides to form uncoated superparamagnetic iron oxide particles.The precipitate is then collected by centrifugation or application of amagnetic field followed by decantation of the liquid phase.

The gel is then peptized to form a dispersoid by mixing with either 1molar aqueous tetramethylammonium hydroxide (to form an alkalinedispersoid) or 2 molar aqueous perchloric acid (to form an acidicdispersoid) followed by centrifugation and redispersion in water. Bothof these dispersoids show remarkable stability and, being colloidal innature, will not possess large solid particles. The counterions, eithertetramethlylammonium hydroxide or perchlorate, are charged in basic oracidic media, respectively and, thus, prevent complex coagulation insolution; the particles (complexes of iron oxide/counterions) can berepeatedly precipitated and re-dispersed in solution and will retainthis property.

In an alternative embodiment the particles can be collected by theapplication of an external magnetic field rather than centrifugation.The resultant magnetic cake is then peptized by the appropriatecounterion.

The ratio of Fe³⁺ /Fe²⁺ is preferably maintained at about 2/1, but canbe varied between about 0.5/1 and about 4/1. Decreasing the ratio willproduce larger and increasing the ratio will produce smaller sizedparticles. Using the 2/1 ratio and 0.7M NH₄ OH, the average particlesize produced is about 1200 angstroms as measured by light scattering.

6.2. Use of the Particles as MR Imaging Agents 15 The magnetic materialsdescribed above can be used as contrast-enhancing agents for in vivo MRimaging. In one embodiment, the particles are dispersed in a suitableinjection medium, such as distilled water or normal saline, or any otherphysiologically acceptable carrier known in the art, to form adispersoid which is introduced into the subject's vascular system byintravenous injection. The particles are then carried through thevascular system to the target organ where they are taken up.

When intravascularly administered, the particles will be preferentiallytaken up by organs which ordinarily function to cleanse the blood ofimpurities, notably the liver, spleen, and lymph nodes, and the otherorgans which tend to accumulate such impurities, notably bone and neuraltissue and to some extent, lung. In each of these organs and tissues,the uptake into the reticuloendothelial cells will occur byphagocytosis, wherein the particles enter the individual cells inmembrane-bound vesicles; this permits a longer half-life in the cells,as such membrane-bound particles will not tend to clump or aggregate(aggregates are rapidly metabolized and cleared from the organ/tissue).Other uptake mechanisms are possible, e.g., pinocytosis. Also, it ispossible that the other cells of the liver (hepatocytes) may absorb themagnetic particles.

Because cancerous tumor cells can lack the ability of phagocytic uptake,the intravascularly administered particles can serve as valuable toolsin the diagnosis of cancer in the above-mentioned organs, as tumors willbe immediately distinguishable on any image obtained.

In a another embodiment, the particles are administered as dispersoidsin a physiologically acceptable carrier, such as distilled water, intothe gastrointestinal tract, which includes the esophagus, stomach, largeand small intestine, either orally, by intubation, or by enema, in asuitable medium. The particles are preferentially absorbed by the cellsof the tract, especially those of the intestine and, like theintravascularly introduced particles, will exert an effect on T₂ of theorgan or tissue. In this manner, cancers and other debilitating diseasesof the digestive system such as ulcers can be diagnosed and affectedareas pinpointed.

Regardless of the route, once administered, the particles distribute toand collect rapidly in the target organs, generally in 30-minutes to anhour.

In the organ, these superparamagnetic particles will alter the magneticfields produced by the MR imager. These altered fields will exert aneffect on the magnetic properties of the hydrogen nuclei (protons) inneighboring molecules; notably affected is the spin-spin relaxationtime, T₂. This parameter is shortened which can result in imagedarkening. Thus, the contrast is enhanced between areas which absorb theparticles rapidly and those which absorb them slowly or not at all.

The particles are however, ultimately biodegradeable and the iron can beutilized by the body for physiological requirements. The contrast effectwill vary with the dose, being longer at higher doses, and also with theorgan imaged. Particularly in the liver and spleen (which store iron forphysiological use) the effect can be observed for 14 days or more, (seeSection 7.6), and, often, as long as 30 days.

The localization in these organs, which store iron for ultimateincorporation into hemoglobin, reveals that the iron oxide particleswill ultimately serve as a source of metabolizable iron and, in fact,will be incorporated in the subjects hemoglobin. Thus, these materialscan also be useful in the treatment of anemia.

The differences in parameter values are interpreted by computer and usedto generate an image of the organ in question. In the cases, asmentioned above, where uptake occurs by phagocytic processes (notablythe liver, spleen, lymph nodes, and bone and neural tissue and to someextent, lung) such an image will clearly and distinctly differentiatebetween cancerous and healthy tissue, allowing for tumor location. Inother organs and/or in the diagnosis of other diseases, modifications ofthe coating of these particles by the attachment of various functionalgroups will stimulate uptake by the organ or cell of choice. For exampleantibodies to a particular tumor cell (e.g. lung carcinoma) can beattached to the surface of a coated particle, stimulating uptake by thatorgan if such a cell is present. In this way, the method can serve adiagnostic tool for many diseases.

6.3. Preparation of Superparamagnetic Fluids

The superparamagnetic fluids useful as imaging agents in this inventionare preferably prepared in a three step process which comprises thesteps of: formation of a superparamagnetic metal oxide; oxidation anddispersion of this oxide by sonication; and dialysis in buffer. Thisprocess yields stable biodegradable superparamagnetic metal oxides thatowe their stability primarily to their anion retaining properties. Themetal oxides may be uncoated or associated to organic polymericsubstances. Each of these steps is discussed separately below.

6.3.1. Formation of Superparamagnetic Metal Oxide

Formation of the superparamagnetic metal oxide is accomplished by mixingthe appropriate metal salts with a base. In a preferred embodiment, thisis accomplished by mixing an aqueous solution or suspension of divalentand trivalent iron salts (FeCl₂ /FeCl₃) with a base such as sodiumhydroxide (NaOH). In addition, metals similar in structure to iron, suchas Co and Mn, can be incorporated into the ultimate superparamagneticfluid by replacing a portion, preferably 1/2 or less, of the divalentiron salt with a divalent salt of that metal. The result is when a mixedmetal oxide preciptate containing both ferrous and ferric oxides, aswell as oxides of the divalent metal.

When iron salts are used the ratio of Fe³⁺ /Fe²⁺ can be varied from 1/4to 4/1 and still produce usable product. Thus, a wide range of saltmixtures can be utilized.

Once the salts are mixed with the base, a superparamagnetic metal oxideprecipitate is formed. The use of a high concentration of reactants andan abrupt change in pH favors the formation of small superparamagneticmetal oxides. Such oxides are preferred for use in the subsequent stepsof this process.

6.3.2. Dispersion and Oxidation

In the second process step, the superparamagnetic metal oxide preparedin 6.3.1. is dispersed and oxidized further by sonication. Thesonication, which can be conducted at ambient or elevated temperatures(up to 100° C.) serves a dual purpose: it serves to disperse anyclusters of superparamagnetic particles (which increases the ultimateeffects of the material on proton relaxation and, hence, enhances theireffectiveness as MR contract agents) and, additionally, it serves tooxidize most, if not all, of ferrous oxide (Fe²⁺) to ferric oxide(Fe³⁺). The resultant material, remarkably, is a solublesuperparamagnetic iron oxyhydroxide which forms a superparamagneticfluid.

The sonication can be accomplished in any commercial apparatus includingin a continuous flow sonicator or by a sonic probe. The former isespecially useful when large volumes of materials are being handled and,for a continuous process, can be coupled with a heating and coolingapparatus to permit heating of the iron oxide prior to or aftersonication (to increase the dispersion and oxidation of the oxides ) andsubsequent cooling of the sonicated mixture to facilitate collection.

6.3.3. Dialysis

The final step in the process is the transfer of the solution to anaqueous polycarboxylic buffer suitable for in vivo use. This transfer isaccomplished by dialyzing the fluid against the buffer at neutral pH,generally at a pH of 6-9, preferably 6-8.3. These conditions result in astable superparamagnetic fluid; under acidic conditions (below a pH ofabout 6) a significant amount of a chelate of the iron begins to formrather than the superparamagnetic iron oxide.

In the process, the fluid from 6.3.2. is centrifuged, to remove largeroxide aggregates, and the supernatant is dialyzed against the buffer.The preferred buffer contains a citrate salt, because of its suitabilityfor in vivo use and its long history as an injectable agent, but ingeneral buffers containing salts of any polycarboxylic acid (such astartrate, succinate) or maleatic buffers allow for the formation ofstable superparamagnetic fluids. The resultant fluids can then beautoclaved and stored until needed.

6.3.4. Preparation of Stable Superparmagnetic Fluids Containing MetalOxides Associated WITH Organic Polymeric Substances

Superparamagnetic fluids containing metal oxides to which organicpolymeric substances are associated can be prepared by modifications ofthe above procedure. Such organic polymers or coatings can be selectedfrom a wide array of polymeric materials including, but not limited to,carbohydrates such as dextran (preferably having a molecular weightbetween 5,000 and 250,000 daltons), proteins (preferably having amolecular weight between 5,000 and 250,000 daltons) such as bovine orhuman serum albumin, polypeptides (preferably having molecular weightsbetween 5,000 and 250,000 daltons) such as polylysine andpolyglutamates, and polymerizable (preferably to a molecular weightbetween 5,000 and 250,000 daltons) organosilanes such asN-2-aminoethyl-3-aminopropyltrimethoxysilane. Briefly, attachment orassociative procedure the attachment can be accomplished during eitherthe first or the second steps.

When the procedure is accomplished during the first step, the polymericmaterial is mixed with the salt solution prior to the supermagneticmetal oxide precipitation. The polymeric material is associated to theresultant precipitate, and remains associated during the subsequentsteps. Any unbound coating or polymeric agent is removed during dialysis(step 3). In a preferred embodiment, superparamagnetic fluids containingdextranized iron oxides can be formed in this manner.

The procedure can also be performed during the dispersion and oxidationsecond step by adding the polymeric substance prior to the sonicationand subsequently sonicating the mixture to form the correspondingoxyhydroxide. Again, the unbound polymeric agents may be removed bydialysis.

Superparamagnetic fluids containing silanized iron oxides are preparedin a similar manner. Initially, the iron oxides are subjected tosonication to form the oxyhydroxides. The organosilane is then added andthe mixture is sonicated to disperse the materials. Finally, the silaneis or associated to the surface via a dehydration reaction. Thepolymerization of the silane may occur before or after the deposition onthe oxyhydroxide surface.

In one embodiment, the silanization reaction occurs in two steps First,a trimethoxysilane is placed in the sonicated mixture which condenses toform silane polymers: ##STR1##

The mixture is then sonicated, after which these polymers associate withthe metal oxide, presumably by forming a covalent bond with surface ORgroups through dehydration: ##STR2## Adsorption of silane polymers tothe metal oxide is also possible.

An important aspect of this procedure is the method of dehydration usedto effect the adsorptive or covalent binding of the silane polymer tothe metal oxide. This association is accomplished by heating the silanepolymer and metal oxide in the presence of a wetting agent miscible inboth the organic solvent and water. Glycerol, with a boiling point ofabout 290° C., is a suitable wetting agent. Heating to about 105° C. inthe presence of glycerol serves two purposes. It insures the evaporationof water, the organic solvent (which may be e.g., methanol, ethanol,dioxane, acetone or other moderately polar solvents) and any excesssilane monomer. Moreover, the presence of glycerol prevents theaggregation or clumping and potential cross linking of particles that isan inherent problem of other silanization techniques known in the artwherein dehydration is brought about by heating to dryness. Thus, fewlarge aggregates are formed. Any large aggregates are removed bycentrifugation and the unbound silane is removed by hydrolysis.

b 6.3.5. Advantages of the Superparamagnetic Fluid Preparation Process

The process used to prepare the superparamagnetic fluids of thisinvention is uniquely suited for preparing magnetic fluids suitable forin vivo use. Specifically, the following advantages are noted:

1. At no time is the material dried, nor is it precipitated after theintial formation of the superparamagnetic oxides. Such operations bringparticles into close proximity with each other resulting in clusteringand aggregation, which adversely affects their utility as MR contrastagents. Further, at no time are the metal oxides removed fromsuperparamagnetic fluid by precipitation or filtration; in fact, theycannot be so removed. In dilute concentrations, the metal oxides willpass through a 0.22 micron filter.

2. Because the material is never precipitated (after the initialformation of the iron oxide), acids or bases are not needed toresolubilize the iron oxide. Use of acids tends to dissolve iron oxides,yielding ferric ion which is toxic and must be removed prior to in vivouse. Strong bases are also poorly suited for use in the preparation ofpharmaceutical solutions of superparamagnetic fluids. Strong bases canhydrolyze biological molecules attached to iron oxides such as proteinsor polysaccharides. Amine-containing strong bases can react withpolysaccharides in the well known Malliard reaction.

3. Changes in solvents, such as to citrate buffer, are accomplished bydialysis. Many other methods (such as that described in U.S. Pat. No.4,001,288) of iron oxide preparation require removal of iron oxides fromsolution to accomplish changes in solvent, often using acid or base toresolubilize the precipitate.

4. The attachment of coating materials to the particles during thepreparation permits a wide array of biologically active molecules suchas antibodies, antigens, serum proteins or other materials to beattached. The attached biologically active molecule can serve to directthe superparamagnetic agent in vivo, as described in section 6.5.

b 6.4 Characterstics of Superparamagnetic Fluids 6.4.1. MagneticProperties

The fluids produced by the methods described in section 6.3. arecharacterized by a high magnetic moment in a high magnetic field(generally, about 5 to about 90 EMU/gm of metal oxide) and a negligiblemagnetic moment in the absence of an applied field (i.e., a magneticsquareness of less than 0.1). Such behavior is characteristic ofsuperparamagnetic particles.

FIG. 4 shows magnetic hysteresis loops for typical paramagnetic,superparamagnetic and ferromagnetic iron oxides. Magnetization wasmeasured in a vibrating sample magnetometer with up to 6,000 Gauss, 25°C. At nigh magnetic fields, the superparamagnetic fluid of the inventionis nearly as magnetic as ferromagnetic iron oxide and far more magneticthan the paramagnetic ferric oxyhydroxide. Also, the solutions of theinvention are superparamagnetic rather than ferromagnetic, losingvirtually all of their magnetic moment in the absence of an appliedmagnetic field. In fact, the superparamagnetic solutions of thisinvention are characterized by a saturation magnetization of 30 EMU/gmor greater, with the loss of more than 90% of this magnetism in theabsence of a magnetic field.

6.4.2. Retention of Citrate and Stability of Superparamagnetic Fluids

The retention of citrate (from aqueous sodium, potassium, or ammoniumcitrate buffer) can be used to distinguish the superparamagnetic fluidsof this invention from other iron oxides. Studies of citrate bindingcapacity of commercially available forms of iron oxides and ferricoxyhydroxides reveals that the iron oxides in the superparamagneticfluids of this invention are capable of retaining nearly as much citrateas the paramagnetic (ionic) ferric oxyhydroxide, while gamma ferricoxide and magnetite cannot retain significant amounts of citrate. Theinability of iron oxides to retain citrate, coupled with the ability offerric oxyhydroxide to do so, strongly suggest that citrate does notadsorb to the surfaces of iron oxides prepared according to the methodof this invention (see Section 6.3) through the usual chemicaladsorption mechanism. The retention of anions like citrate by thesuperparamagnetic iron oxides of the invention indicates these materialshave an ionic character similar to the ferric oxyhydroxides.

The stability of fluids of the invention is shown in FIGS. 5A and 5Bwhere a superparamagnetic fluid made according to the proceduredescribed in section 6.3. was subjected to autoclaving with and withoutcitrate. Addition of 50 mM citrate stabilized a solution of 1.26M ironoxide, preventing the gelation of the material.

The stability of the oxyhydroxide solutions of iron (i.e., thesuperparamagnetic fluids) is related to the exchange of hydroxide forcitrate ion. Both paramagnetic and superparamagnetic oxyhydroxidesretain citrate in a similar fashion:

    3 FeO:OH+Citrate.sup.3- →(FeO).sub.3 --Citrate+3 OH.sup.-

Instead of trying to block the Van der Waals forces between neutralcrystals with polymers, by attaching surfactants, or forming complexes,the general approach used by others in forming ferrofluids, theinvention's superparamagnetic fluids are stabilized due to the ioniccharacter of the iron oxide and the choice of appropriate anions.

The stable solutions of this invention comprise a metal concentrationranging from 0.05 to 5 molar and a citrate ion concentration of 0.001 to0.1 moles of citrate/mole preferably 0.01 to 0.1 moles of citrate/moleof iron at a pH ranging from about 6 to about 10. As the concentrationof iron in the solution is increased, the ratio of citrate/iron mustalso increase to yield the stability. Thus, they are compatible withphysiological conditions.

The superparamagnetic fluids of the invention owe their stability insolution, not to their coating with polymers or surfactants, but to theexistence of a cationic character of the iron oxide and itsstabilization with anions such as citrate. In general, polymericcoatings, though they help stabilize iron oxides, are not sufficient toprotect them against the rigors of autoclaving. In contrast,superparamagnetic fluids made according to the invention can be madeomitting the polymer altogether and are highly stable.

6.4.3. Effectiveness as MR Contrast Agents

In evaluating magnetic materials as MR contrast agents, the ability ofmaterials to shorten proton relaxation time can be more important thanbulk magnetic properties such as magnetization. Since MR imaging worksby determining the rates of two types of proton relaxations in varioustissues and, by using variations in those relaxation rates, develops animage, the differences in proton relaxation times between the tissuesmust be sufficiently great to obtain a good quality image. As statedsupra, MR contrast agents work by shortening proton relaxation time and,thus, increases the contrast and overall image quality. Two relaxationparameters termed spin-spin relaxation time (T₁) and spin-latticerelaxation time (T₂) are used in the generation of the MR image.

In experiments evaluating the effect of these materials as contrastagents, it was found that the superparamagnetic fluids have a muchgreater effect on both T₁ and T₂ than any commercially available ironcompounds including chelated ferric ion, paramagnetic ferricoxyhydroxides, gamma ferric oxides, and superparamagnetic iron oxideclusters Seer U.S. Pat. No. 4,584,088. ln fact, the material of theinvention is remarkable in its ability to shorten proton relaxation. Thematerials prepared according to the invention are more potent enhancersof proton relaxation than either ferromagnetic materials or paramagneticferric oxyhydroxide. In addition, the highly dispersed state of thematerials of the invention produces higher relaxivities than thosecharacteristic of clustered materials (See 7.12 and Table III). Theprocess, thus, yields superparamagnetic solutions optimized for theireffects on proton relaxation time.

The high relaxivity (see Table III) of the materials of the invention isimportant to their pharmaceutical use as MR contrast agents, because itresults in large effects on the MR image with small doses of iron. Forexample, superparamagnetic iron oxides made according to the inventioncan profoundly improve liver imaging at doses of 1 mg of iron perkilogram of rat, while the LD50 for the rat is greater that 250 mg ofiron per kilogram.

6.5. Biodegradability

Both the superparamagnetic particles in the dispersoids and the metaloxides in the superparamagnetic fluids of the invention have been foundto be biodegradable when administered in vivo (see Examples 7.6 and7.15). In fact, iron, the predominant species in the dispersoids andfluids accumulate in the liver, where it is eventually catabolized andincorporated into the subject's hemoglobin. Thus, the dispersoids andfluids can be used in the treatment of anemia, and indeed, the fluidshave been shown to be as effective as Imferon (a commercially usedpreparation for treatment of anemia in humans) in the restoration ofnormal hematocrit levels in anemic rats.

6.6. Directability

Both the superparamagnetic particles in the dispersoids and the metaloxides in the superparamagnetic fluids of the invention can be coatedwith various coating agents as described supra. The use of such coatingspermits the attachment of various biological molecules to the imagingagents to permit targeting of various organs. For example, antibodiescan be bound by a variety of methods including diazotization andattachment through a glutaraldehyde or carbodiimide coupling moiety(Examples of these coupling methods can be found in U.S. Pat. No.4,628,037, which is incorporated herein by reference). Use of methodssuch as these permits maximum flexibility, as an antibody-directedsuperparamagnetic metal oxide can bind to a specific type of cell ortissue. This can permit an image to be generated which differentiatesbetween the target and the surrounding tissue.

In addition to antibodies, other biological molecules which affectdirectability can also be attached to the particles as the particularapplication dictates. Some possible applications are listed below:

    ______________________________________                                        Antibodies        Application                                                 ______________________________________                                        1.     Anti-heart myosin                                                                            Imaging infarcted area                                                        of heart                                                2.     Anti-fibrin    Image clot                                              3.     Anti-T-cells   Lymphoma                                                4.     Anti-CEA       Colonic tumor imaging                                   5.     Anti-melanoma  Melanoma imaging                                               antibodies                                                             6.     Anti-ovarian   Ovarian cancer imaging                                         cancer antibodies                                                      7.     IgG            Fc receptor                                                                   delineation                                             ______________________________________                                    

Carbohydrates

1. Bacterial lipopolysaccharides

2. Cellulose

3. Mucopolysaccharides

4. Starch

5. Modification of carbohydrate after synthesis, e.g., dextran coatingmade positively or negatively charged, diethylamino (DEAE) cellulose orcarboxymethyl (CM) dextran.

    ______________________________________                                        Hormones           Application                                                ______________________________________                                        1.     Insulin         Insulin receptor                                                              status as in maturity                                                         onset diabetes                                         2.     Thyroid stimulating                                                                           Thyroid disease                                               hormone                                                                3.     Acetylcholine   Delineation of                                                (or analogs)    neural receptors                                       4.     Serum low density                                                                             Delineation of                                                lipoprotein     familial hyper                                                                cholesterolemia                                        5.     Hormone analogs Delineation of                                                including drugs endocrine system and                                                          receptors                                              6.     Serum transferrin                                                                             Transferrin receptors                                                         delineation                                            ______________________________________                                    

6.7 Method for Extending the Serum Lifetime Of An MR Image ContrastAgent

To extend the lifetime of an MR imaging agent in the serum of a subject,if desired, it is necessary to prevent its absorption by thereticuloendothelial system (RES). It has been found that this can beaccomplished by introducing to the subject a blocking agent whicheffectively competes with the imaging agent for binding the RESreceptors responsible for removing the MR contrast agent from thebloodstream. There are a number of phagocytic receptors which functionindependently of each other. As a result, no single material is equallyeffective at blocking all the RES receptors and any blocking agent mustbe specific for the imaging agent. (See Davis et al. in "PolymericNanoparticles and Microspheres", Gurot, P. and Covreur, P., eds, (CRCPress, 1986) p. 180).

In the procedure, the subject is given a dose of paramagnetic iron oxideeither prior to or along with the administration of the imaging agent.For the best results, the paramagnetic iron oxide should be as simlar tothe agent as practical especially in particle size and coating. After ashort time interval, generally 15-20 minutes during which time theparamagnetic material circulates in the bloodstream and binds to the RESreceptors, the imaging agent is administered. By proper choice of theparamagnetic dosage, the lifetime of the imaging agent in the serum isgreatly enhanced.

An excellent blocking agent for a superparmagnetic MR agent is aparamagnetic form of the same material. This is because theeffectiveness of a blocking agent depends on whether a competition forreceptors results; cell surface receptors bind materials in circulationprior to internalization. This internalization is termed pinocytosis(removal of liquids) or phagocytosis (removal of particles). Ifcompetition is created, which blocks removal of the superparamagnetic MRcontrast agent, the removal of the contrast agent will be hindered.Because the RES receptors are specific and will bind substances of onlyone particular size or shape, this competition is best observed amongmaterials which are physically similar. Since a paramagnetic particlecan differ from a superparamagnetic particle only in its core magneticproperties and rather than its surface chemistry, a high degree ofcompetition is inevitable and, thus, the paramagnetic material is ahighly efficient blocking agent.

In a preferred embodiment, dextran-coated paramagnetic iron oxide isused as a blocking agent for dextran coated superparamagnetic ironoxide. This material is ideal as a blocking agent for dextran-coatedsuperparmagnetic iron oxide contrast agents for the reasons below:

1. Its effect on proton relaxation is virtually undectectable by MR.

2. It can be made by the process used for superparamagnetic materials,but without the use of divalent iron, (which is required for thesuperparamagnetic product). The MR contrast agent and blocking agent areidentical except for the fine structure of the iron- oxide whichdetermines magnetism and its effect on proton relaxation. From the pointof view of the cell surface receptor governing removal from circulationthe superparmagnetic imaging agent and blocking agent are identical.

3. It is non-toxic in humans and has an established therapeutic use inthe treatment of anemia. In fact, therapeutically approved paramagneticdextran (Imferon) can be used as a blocking agent for thesuperparmagnetic MR contrast agents of the invention.

The extension of serum lifetime is of particular importance when MRmeasurements are used to confirm blood circulation (or lack thereof). Insuch measurements, the contrast agent is introduced parenterally andpermitted to circulate. By measurement of T₁ and T₂, presence or absenceof blood circulation can be determined. Such a procedure can be avaluable tool in the diagnosis of blood circulation disorders, and canbe used to detect the flow of blood into areas where it is normallyexcluded, such as in strokes.

7. Examples 7.1. Preparation of Dextran-Coated Particles

To a solution of 500 mls of 0.28M FeCl₃, 0.16M FeCl₂ and 12.5% w/vdextran, (molecular weight 71,000 daltons from Sigma Chemical Company,Cat. #D1390) is added 500 mls 7.5% NH4OH over a 2 minute period. Ablack, magnetic solid forms comprised of large and small particles. Thematerial is stirred for 5 minutes and then heated for 30 minutes at 70°C. The solution is centrifuged for 1500×g for 15 minutes to remove largeparticles, and the small particles are dialyzed against 10 gallons of H₂O for three days, changing the water each day.

The resultant particles exhibit a diameter of about 1400 angstroms ameasured by light scattering.

7.2. Preparation of Particles Coated with Bovine Serum Albumin

To a solution of 80 mls of 0.5% bovine serum albumin (BSA), 0.27M FeCl₃,and 0.16M FeCl₂, is added 80 mls of 7.5% NH₄ OH. A black, magnetic solidforms comprised of particles. The mixture is allowed to stand for 5minutes and then centrifuged at 1,500 ×g for 15 minutes to remove largerparticles. The pellet is discarded and the supernatant placed in adialysis bag and dialyzed against 3 changes of 10 gallons of distilledwater. Larger particles are again removed by centrifugation as above anddiscarded. Particles are then concentrated by ultrafiltration using anXM-50 membrane and a stirred cell filtration device from AmiconCorporation, Lexington, MA.

The resultant particles exhibit a diameter of about 1730 angstroms asmeasured by light scattering.

7.3. Preparation of Uncoated Particles

One hundred milliliters of solution of 0.8M FeCl₃, 0.4M FeCl₂ and 0.4MHCl is added dropwise to 1000 ml of 2.4% NH₄ OH and mixed for 5 minutes.A black, magnetic solid forms comprised of easily visible particles. Forparticles to be visible, they must be larger than the wavelength ofscattered light which is about 500 nm (0.5 microns). The particles areisolated by attracting them to a permanent magnet on the outside of thereaction vessel and the solution decanted. To the magnetic cake is added55 mls of 50% triethylamine in water. Smaller particles are created. Themixture is dialyzed overnight against water which causes the largeparticles to reappear. Just enough triethylamine is then added to againcreate the smaller particles resulting from the addition oftriethylamine. The particles are then filtered through a 0.2 micronfilter indicating the final material is below this size.

7.4. Use of Particles In Liver Tumor Visualization

The effect of the dextran-coated particles of Section 7.1. on the imageof a rat liver tumor is demonstrated in FIG. 2, which presentsreproductions of five images obtained on a Technicare MR imager. Theimages in FIGS. 2A and 2B were obtained prior to the introduction of theimaging agent using different imager settings, in neither case can thetumor be clearly seen; FIGS. 2C and 2D are images of the same 15 ratliver and were obtained after a single 0.5 mg/kg dose of the Section6.1. dextran-coated particle by intravenous injection through the tailvein, the tumor is easily seen and the overall size and shape can begauged; in FIG. 2E the tumor is marked by cross-hairs to aid invisualization.

7.5. Comparative Effect of Superparamagnetic Particles And FerromagneticParticles On T2

FIG. 1 compares the T₂ of agar gel in the presence of dextran-coatedparticles (produced in Example 7.1.) and the ferromagnetic particlePf-2228 (Pfizer). The relaxation times in the presence of varyingconcentrations of each particle were determined on an IBM PC-20 NMRspectrometer at 0.47 Tesla (4700 Gauss). It can clearly be seen that thesuperparamagnetic particle produces a much greater effect on T₂ than theferromagnetic particle. Given the fact that superparamagnetic materialsare much less magnetic than ferromagnetic materials, this result isquite surprising.

b 7.6. Biodegradability of Dextran-Coated Particles

A dispersion of uncoated superparamagnetic iron oxide particles in waterwas intravenously injected into Sprague-Dawley rats at a dosages 20, 37and 243 micromoles Fe/kg of body weight. Periodically, the rats weresacrificed, and T₂ of the liver tissue was measured on an IBM PC-20 NMRSpectrometer. The results are presented in FIG. 3.

The data demonstrate that T₂ undergoes a marked drop rapidly after theinjection, and then begins to recover slowly, presumably as the iron ismetabolized. However, the effects are still detectable even two weeksafter administration. Also, the effects are more marked with the higherdosages. Thus the particles have an extended lifetime in these organs.The liver and spleen are the major organs which store iron forincorporation into hemoglobin, and, indeed these materials areultimately incorporated into the hemoglobin of the rat.

7.7. Biodistribution of BSA-Coated Particles

Six Sprague-Dawley rats of about 200 gm each were injected intravenouslywith 0.4 mg of the BSA-coated particle (produced in Section 7.2.) indistilled water. Two rats each were sacrificed at 90 minutes, 24 hours,and 7 days after injection and the relaxation times (T₁ and T₂ ofvarious organs were measured on an IBM PC-20 NMR Spectrometer. Thefollowing results were obtained:

                  TABLE I                                                         ______________________________________                                        DISTRIBUTION OF BSA-                                                          COATED PARTICLE IN RAT ORGANS AND TISSUES                                     Time After   Relaxation Times (msec)                                          Injection    Liver   Spleen    Lung  Blood                                    ______________________________________                                        Control T.sub.1  0.279   0.544   0.688 0.786                                  N.sup.1 = 6                                                                           T.sub.2  32      48.3    57    158                                    90 min  T.sub.1  0.232   0.396   0.656 0.901                                  N = 2   T.sub.2  20      22      56    136                                    24 hours                                                                              T.sub.1  0.279   0.494   0.735 1.084                                  N = 2   T.sub.2  22      44      68    155                                    7 days  T.sub.1  0.268   0.572   0.712 0.972                                  N = 2   T.sub.2  31      49      68    162                                    ______________________________________                                         .sup.1 N is the number of rats examined.                                 

The data suggest that both the blood and the lung rapidly clear themagnetic material exhibiting nearly no effect on the relaxation times 50minutes after the injection. The spleen demonstrates a moderately rapidrecovery, exhibiting a substantial reduction in both T₁ and T₂ 90minutes after the injection, but nearly no residual recovery rates. T₁attains its original value after 24 hours, while T₂ remainssubstantially reduced after 24 hours and exhibits recovery after 7 days.

7.8. Comparative Biodistribution of Uncoated and Dextranized Particles

In this experimental series, the biodistribution of b three uncoated andfour dextran-coated particles was examined. The uncoated agents wereproduced according to the procedure of Section 7.3., the dextran-coatedparticles were produced according to the procedure of Section 7.1.except that the molecular weight of the dextran used for the coating wasvaried (see TABLE II). Prior to each experiment, the contrast agentswere dialyzed against distilled water and subsequently injected intoseparate groups of Sprague-Dawley rats in a distilled water carrier. Therats were periodically sacrificed and the relaxation times of the liver,spleen, and lung were determined on an IBM PC-20 NMR Spectrometer.Preprogrammed inversion recovery and Carr, Purcell, Meiboom, Gill pulsesequences were used to determine T₁ and T₂, respectively.

The results were as follows:

                  TABLE II                                                        ______________________________________                                                                       Relaxation Times                               Com-  Coat-            Time After                                                                            (msec.)                                        plex  ing     Dose     Dose    Liver Spleen                                                                              Lung                               ______________________________________                                        Con-  --      None     --    T.sub.1                                                                           0.27  0.54  0.717                            trol                         T.sub.2                                                                           32    48    64                               AMI-  None    24.2     2.5 hr.                                                                             T.sub.1                                                                           0.222 0.420 0.626                            12            μmoles/     T.sub.2                                                                           22.7  26.0  45.8                                           kg                                                                                     18 hr.                                                                              T.sub.1                                                                           0.254 0.532 0.752                                                         T.sub.2                                                                           29.6  42.9  68.2                                                    1 wk. T.sub.1                                                                           0.239 0.528 0.730                                                         T.sub.2                                                                           31.6  43.8  72.0                                                    2 wk. T.sub.1                                                                           0.240 0.462 0.702                                                         T.sub.2                                                                           29.4  35.5  79.5                             AMI-  None    24.6     2.5 hr.                                                                             T.sub.1                                                                           0.221 0.424 0.672                            13            μmoles/     T.sub.2                                                                           16.9  28.0  65.2                                           kg                                                                                     18 hr.                                                                              T.sub.1                                                                           0.218 0.386 0.802                                                         T.sub.2                                                                           18.8  29.0  80.8                                                    1 wk. T.sub.1                                                                           0.236 0.443 1.753                                                         T.sub.2                                                                           26.0  38.5  80.4                                                    2 wk. T.sub.1                                                                           0.236 0.493 0.722                                                         T.sub.2                                                                           28.2  43.8  80.8                             AMI-  None    25.4     2 hr. T.sub.1                                                                           0.238 0.470 0.706                            14            μmoles/     T.sub.2                                                                           20.8  31.8  72.4                                           kg                                                                                     18 hr.                                                                              T.sub.1                                                                           0.238 0.436 0.750                                                         T.sub.2                                                                           20.4  34.7  69.6                                                    1 wk. T.sub.1                                                                           0.216 0.522 0.755                                                         T.sub.2                                                                           26.7  41.7  80.4                                                    2 wk. T.sub.1                                                                           0.227 0.452 0.698                                                         T.sub.2                                                                           24.8  43.6  78.7                             AMI-  Dex-    36.8     4 hr. T.sub.1                                                                           0.238 0.300 0.672                            15    tran    μmoles/     T.sub.2                                                                           17.8  19.4  56.4                                   9,000   kg                                                                                     24 hr.                                                                              T.sub.1                                                                           0.253 0.387 0.740                                                         T.sub.2                                                                           21.1  26.4  73.2                                                    1 wk. T.sub.1                                                                           0.219 0.485 0.766                                                         T.sub.2                                                                           25.6  36.7  78.1                                                    2 wk. T.sub.1                                                                           0.258 0.523 0.718                                                         T.sub.2                                                                           28.7  39.1  69.9                             AMI-  Dex-    32.4     4 hr. T.sub.1                                                                           0.248 0.302 0.678                            16    tran    μmoles/     T.sub. 2                                                                          18.8  16.5  56.2                                   17,900  kg                                                                                     24 hr.                                                                              T.sub.1                                                                           0.238 0.384 0.703                                                         T.sub.2                                                                           19.9  24.9  71.6                                                    1 wk. T.sub.1                                                                           0.197 0.470 0.725                                                         T.sub.2                                                                           25.3  37.1  74.6                                                    2 wk. T.sub.1                                                                           0.258 0.525 0.731                                                         T.sub.2                                                                           28.9  44.8  73.3                             AMI-  Dex-    33.1     4 hr. T.sub.1                                                                           0.244 0.318 0.674                            17    tran    μmoles/     T.sub.2                                                                           16.0  17.4  54.4                                   35,600  kg                                                                                     24 hr.                                                                              T.sub.1                                                                           0.247 0.388 0.690                                                         T.sub.2                                                                           20.2  22.9  76.4                                                    1 wk. T.sub.1                                                                           0.214 0.500 0.696                                                         T.sub.2                                                                           24.3  44.0  76.0                                                    2 wk. T.sub.1                                                                           0.244 0.562 0.726                                                         T.sub.2                                                                           28.6  48.6  70.6                             AMI-  Dex-    39.2     4 hr. T.sub.1                                                                           0.228 0.237 0.526                            18    tran    μmoles/     T.sub.2                                                                           20.0  17.7  58.6                                   249,000 kg                                                                                     24 hr.                                                                              T.sub.1                                                                           0.238 0.354 0.654                                                         T.sub.2                                                                           21.0  22.0  68.2                                                    1 wk. T.sub.1                                                                           0.235 0.492 0.645                                                         T.sub.2                                                                           31.4  36.1  71.3                                                    2 wk. T.sub.1                                                                           0.240 0.52  0.748                                                         T.sub.2                                                                           31.0  39.8  71.3                             ______________________________________                                    

As before, the data suggest that the contrast agents are rapidly clearedfrom the lung, and are longer lived in the spleen and the liver.Additionally, it can be seen that the dextran-coated particles arecleared less rapidly than the uncoated ones, exerting a significanteffect on the T₂ values of the liver and spleen for about one week.

7.9. Preparation of Superparamagnetic Fluids Containing Uncoated MetalOxide 7.9.1 Preparation of Superparamagnetic Iron Oxide

A solution of 0.25M ferrous chloride and 0.5M ferric chloride (600 ml)was poured into a solution.,. of 5M NaOH (600 ml). A black magneticoxide precipitate was formed. This precipitate was washed repeatedly bybase and decanted until a pH of about 9 was achieved.

7.9.2 Disperson and Oxidation

In a beaker, 400 ml of magnetic oxide (about 15 grams) from Section7.9.1 and 25 ml of glacial acetic acid were mixed. A sonic probe wasplaced in the beaker and the solution was sonicated at high intensityfor 2 minutes. The sonic probe was then remove and the solutioncentrifuged at 1,000×g for 20 minutes. The pellet was discarded and thesupernatant liquid was retained.

7.9.3 Transfer to Citrate Buffer

The supernatant, from Section 7.9.2, was dialyzed against ammoniumcitrate buffer by use of a hollow fiber dialyzer/concentrator, model DC2 (AMICON Corp. Danvers, MA). The ammonium citrate buffer is 10 mMcitrate, adjusted to pH 8.2 with NH₄ OH. The result is an autoclavable,homogeneous supermagnetic fluid.

7.10. Preparation of an Aqueous, Stable Superparamagnetic FluidContaining Metal Oxide With Dextran Attached 7.10.1 Synthesis of IronOxide

Five liters of a solution containing 755 g FeCl₃ 6H₂ O and 320 g FeCl₂4H₂ O was added slowly to 5 liters of 16% NH₄ OH containing 2500 gmdextran (MW=10-15,000). The iron salt solution was added over 5 minutesduring which time the base was vigorously stirred during addition.

A black magnetic slurry was formed.

7.10.2. Dispersion, Oxidation and Heating

The 10 liters of slurry (from Section 7.10.1) was pumped through acontinuous flow sonicator connected to a 100° C. heating coil andcooling coil apparatus as indicated in FIG. 6. The pumping rate wasabout 0.4 liters per minute and pumping was continued for about 30minutes. The resultant solution was subsequently centrifuged and theprecipitated pellet was discarded.

7.10.3. Removal of Unreacted Dextran, Transfer to Citrate Buffer andSterilization

The supernatant (from Section 7.10.3) was diluted to a total volume of20 liters with deionized, sterile water and the resultant solution wasdialyzed as in Example 7.9, except that a larger dialyzerconcentrator<the DC 10, was used. The dialyzer cartridge had a 100,000dalton molecular weight cutoff, permitting removal of dextran.Ultrafiltration was accomplished in a noncontinuous fashion, reducingthe volume from 20 to 5 liters and adding 16 liter volumes of solution.Five volumes of 16 liters of deionized, distilled water were added.

Sodium citrate was then added as 1M citrate buffer stock and thesolution was dialyzed as in Example 7.9. The resultant citrate wasadjusted to pH 6.5 with NaOH before autoclaving. The citrate to ironratio was between 0.01 and 0.1 citrate/Fe in the final solution. (Forexample, for an iron concentration of 1.26M, 0.04M citrate was present.The magnetic fluid was bottle d and autoclaved (121° C, 30 minutes). Theresult is a sterile homogenous magnetic fluid as shown in FIG. 5.

7.11. Preparation of an Aqueous Stable Superparamagnetic FluidContaining Metal Oxide with Silane Attached 7.11.1 Preparation of anIron Oxide

A solution of 0.25M ferrous chloride and 0.5M ferric chloride (600 ml)was poured into a solution of 5 M NaOH (600 ml). A black magnetic oxideprecipitate formed which was repeatedly washed by base and decanteduntil a pH of about 9 was achieved.

7.11.2. Dispersion, Oxidation and Silanization

In a beaker 400 ml of magnetic oxide (from Section 7.11.1., about 15grams) and 25 ml of glacial acetic acid were mixed. A sonic probe wasplaced in the beaker and the solution was sonicated at high intensityfor 2 minutes. The sonic probe was then removed and 30 ml ofN-2-aminoethyl-3-aminopropyltrimethoxysilane was added. The resultantmixture was then sonicated as before. The magnetic solution wassubsequently added to 200 ml of glycerol at 50° C. The temperature wasraised to 105° C. and the water was evaporated.

Due to the use of sonication, the material made is far smaller than thatdescribed in U.S. Pat. No. 4,554,088. Due to its small size, it cannotbe manipulated with hand held magnets. The glycerol dehydration step isfrom U.S. Pat. No. 4,554,088 herein incorporated by reference..

7.11.3. Removal of Unreacted Silane and Transfer to Citrate Buffer

The glycerol slurry, from Section 7.11.2, was added to about 800 ml ofwater. Large aggregates of magnetic particles were removed bycentrifuging the slurry at 1,000 ×g for 20 minutes. The supernatant wasthen dialyzed against citrate buffer in a hollow fiber dialysis deviceas in Example 7.9.3.

7.12. Effect of the Superparamagnetic Fluid on Proton Relaxation time

The effects of materials on an in vivo MR image can be evaluated throughthe use of a magnetic resonance spectrometer. In this study, an IBM-PC20 instrument which measures T₁ and T₂ at 25° C., 0.47 Tesla and 20 MHzwas used. Enhancement of proton relaxation can be quantified by takingthe slope of a plot of 1/T, the reciprocal of the relaxation time,versus the concentration of contrast agent. The plot is generallylinear, with the slope being termed the relaxivity and denoted R1 or R2.Relaxivity has units of M⁻¹ sec⁻¹. Higher relaxivity values indicatethat material is more potent per mole of iron at decreasing relaxationtimes of protons and, thus, is a more potent contrast again.Relaxivities for a number of different forms of magnetic materials weredetermined. The following materials were examined:

Superparamagnetic fluid of the invention: A dispersed fluid containingsuperparamagnetic crystals of iron oxide prepared as described inExample 7.10. The magnetization curve of this material is presented inFIG. 4.

Fe₂ O₃ : A ferromagnetic gamma ferric oxide used for data recording.This material was obtained from Pfizer Corp., Minerals, Pigments andMetals Division, catalogue #2228.

Cluster: A silanized cluster of superparamagnetic iron oxide with tensto hundreds of crystals packed into micron-sized particles. Thismaterial was made according to U.S. Pat. No. 4,554,088.

FeO:OH: a paramagnetic, ferric oxyhydroxide used in the treatment ofanemia. It was obtained from Fisons Corporation and is sold under thetrade names of Proferdox (Fisons corporation) or Imferon (Merril DowInc.)

Fe³⁺ /DTPA: a soluble complex of ferric ion anddiethylenetriam:inepentacetic acid (DTPA). (The data for this materialis from Lauffer at al, J. Comp. Assit. Tomog. 9(3), 431 (1985)).

The results were as follows:

                  TABLE III                                                       ______________________________________                                        EFFECT OF DIFFERENT                                                           FORMS OF IRON ON PROTON RELAXATION TIME                                       Material       R1           R2                                                Material       (M.sup.-1 × sec.sup.-1)                                                              (M.sup.-1 × sec.sup.-1)                     ______________________________________                                        superparamagnetic                                                                            4 × 10.sup.+4                                                                        1.6 × 10.sup.+5                             fluid                                                                         gamma Fe.sub.2 O.sub.3                                                                       100          7.2 × 10.sup.+3                             FeO:OH         0            60                                                Cluster        2 × 10.sup.+3                                                                        3 × 10.sup.+4                               Fe.sup.3+ /DTPA                                                                              0.73 × 10.sup.+3                                                                     0.85 × 10.sup.+3                            ______________________________________                                    

Briefly, as the high values R1 and R2 indicate, material of theinvention is remarkable in its ability to shorten proton relaxationtimes For comparison, the value of R2 for ferromagnetic dextranmagnetite is 1.7 ×10⁺⁴ M⁻¹ sec⁻¹ [Ohgushi et al., J. Mag Res. 29, 599(1978)]. This is the highest literature value for R2 of which theauthors are aware. The materials prepared according to the invention aremore potent enhancers of proton relaxation time than eitherferromagnetic materials or paramagnetic ferric oxyhydroxides.

Additionally, well dispersed materials, such as those of the invention,have higher relaxivities than clustered materials. Thus, the process ofthe invention yields superparamagnetic solutions optimized for theireffects on proton relaxation.

7.13. Bulk Magnetic Properties of Superparamagnetic Fluids

Magnetic hysteresis loops were obtained for the samples of the inventionsuperparamagnetic fluid, gamma Fe₂ O₃ (ferromagnetic), and FeO:OH(paramagnetic) examined in Example 7.12, using a commercial vibratingsample magnetometer with fields up to 6000 Gauss, 25° C.. The resultsare presented in FIG. 4.

Briefly, at high magnetic fields, the superparamagnetic fluid of theinvention is nearly as magnetic as ferromagnetic iron oxide and far moremagnetic than the paramagnetic ferric oxyhydroxide, showing a highmagnetic saturation. The fluids of the invention are superparamagneticrather than ferromagnetic, losing virtually all of their magnetic momentin the absence of an applied magnetic field.

7.14. Retention of Citrate

The retention of ¹⁴ C citrate upon dialysis can be used to distinguishvarious forms of iron oxide as shown in Table IV. All iron oxides wereinitially dialyzed against a buffer of 1 mM Tris-Cl, pH 8 before use.Equilibrium dialysis was then performed to determine fraction of citrateretained The concentrations of iron and citrate were 17.8 and 2.6 mM,respectively. The superparamagnetic fluids of the invention retainamounts of citrate similar to commercially available ferricoxyhydroxides indicating that the iron in both preparations is in asimilar chemical form. Commercially available forms of iron oxide, suchas gamma Fe₂ O₃ or Fe₃ O₄, do not retain significant amounts of citrate(the gamma Fe₂ O₃ was the same as that used in Examples 7.12 and 7.13while the Fe₃ O₄ was purchased from Fisher Scientific Inc). Theinability of these commercially available iron oxides to retain citrate,coupled with the ability of ferric oxyhydroxide to do so, stronglysuggests that citrate does not absorb to iron oxide surfaces through theusual chemical adsorption mechanism. The retention of citrate by thesuperparamagnetic iron oxides of the invention indicates these materialshave an ionic character similar to the ferric oxyhydroxides.

                  TABLE IV                                                        ______________________________________                                        RETENTION OF CITRATE                                                          BY SOLUTIONS WITH DIFFERENT IRON OXIDES                                                    Citrate Retained per Iron                                        Material     (mole/mole)                                                      ______________________________________                                        FeO:OH       0.026                                                            Invention    0.019                                                            gamma Fe.sub.2 O.sub.3                                                                     0.0028                                                           Fe.sub.3 O.sub.4                                                                           0.0018                                                           ______________________________________                                    

7.15. Stability of Superparamagnetic Fluids

Superparamagnetic fluids made according to Example 7.10 were subjectedto autoclaving with various concentrations of citrate. At ironconcentration of 1.26M, various concentrations of ammonium citrate, pH8were added, and the resulting solutions heated 1 hour at 121° C. Theresults are presented in FIGS. 5A and 5B. The 6 vials of FIG. 5Bcontained, as shown, citrate concentrations of 100, 50, 25, 15, 10 and 5mM citrate, respectively. The vials were upright during autoclaving butwere placed horizontally for the photograph. With the vials lyinghorizontally, the presence of gelled material is evident when the upperportion of the vial is translucent. The fully blackenend vials (citrateconcentrations between 15 and 100 mM) indicate a solution ofsuperparamagnetic materials was maintained. The two vials on the right(citrate concentration of 5 and 10 mM) show the formation of a gel. FIG.5A further shows the characteristic gel obtained without citrate, orwith inadequate citrate (5 and 10 mM citrate).

7.16. Biodegradability of Superparamagnetic Fluids

Paramagnetic ferric oxyhydroxides are biodegradable and have long beenused for the treatment of anemia. Therefore, the biodegradability of theinvention's superparamagnetic fluids was compared with the paramagneticferric oxyhydroxides. The ability of both iron preparations to reverseanemia in rats was utilized as a model. The paramagnetic ferricoxyhydroxide was Imferon, and has dextran attached. The supermagneticfluid also had dextran attached and was produced as described in Example7.10.

Weanling rats were divided into four groups of five rats each. Rats ingroup 1 received a chow containing iron and were sacrificed at weeks 5,6, 7 and 8 to allow establishment of normal iron (hematocrit) levels inrat tissues. Rats in groups 2, 3 and 4 received an iron deficient diet.Rats in group 2 were also sacrificed at weeks 5, 6, 7, and 8 to allowestablishment of normal iron levels in rat tissues. Rats in groups 2, 3and 4 received an iron deficient diet. After receiving the low iron dietfor 5 weeks, rats in groups 3 and 4 received intravenous (tail vein)injections of iron to reverse their anemia and restore normal levels.Rats in group 3 received Proferdex, while those in group 4 received thedextranized superparamagnetic fluid. Rats receiving iron were injectedwith a single dose of 30 mg of iron per kilogram, a sufficient dose toreverse their anemia. The results are presented in Table V.

                  TABLE V                                                         ______________________________________                                        REVERSAL OF ANEMIA WITH                                                       SUPERPARAMAGNETIC IRON OXIDE PARTICLES                                        Hematocrit (1% red cells in whole blood)                                      week 5        week 6    week 7     week 8                                     avg        sd     avg    sd   avg   sd   avg  sd                              ______________________________________                                        chow    45.1   1.4    45.2 0.5  46.7  0.9  47.0 0.8                           low Fe  28.5   2.6    29.5 2.1  32.7  1.2  34.8 2.3                           Imferon               43.1 1.9  42.8  1.3  46.3 1.2                           Invention             44.3 2.3  42.7  1.1  47.6 1.6                           ______________________________________                                    

It can be seen that the invention's superparamagnetic iron oxiderestores normal hematocrit levels in rats as well as the paramagneticpreparation, Imferon.

7.17. Summary of Superparamagnetic Fluid Properties

The properties of the superparamagnetic fluids of the invention comparedwith solutions made with other types of ferric oxide are summarizedTable VI:

                  TABLE VI                                                        ______________________________________                                        SUMMARY OF PROPERTIES OF AQUEOUS                                              SOLUTIONS OF VARIOUS FERRIC OXIDES                                                   Mag.    MR        Biodegrad-                                                                              Citrate                                           Saturation                                                                            Relaxivity                                                                              ability   retention                                         (FIG. 4)                                                                              (Table II)                                                                              (Table V) (Table IV)                                 ______________________________________                                        FeO:OH   low       none      high    high                                     gamma Fe.sub.2 O.sub.3                                                                 high      some              low                                      Invention                                                                              high      high      high    high                                     ______________________________________                                    

Thus, the superparamagnetic fluids of this invention posess a uniquecombination of magnetic, biological and anion retaining properties.

7.18. Extension of the Serum Lifetime of Dextran CoatedSuperparamagnetic Iron Oxide Particles

To assess the effectiveness of dextran-coated paramagnetic iron oxide asa serum lifetime extender for dextran-coated superparamagnetic ironoxide particles, a comparative study was conducted.

In both trials, a rat of about 300 g was injected with 1 mg Fe/kg bodyweight of dextran-coated superparamagnetic metal oxide produced asdescribed in Example 7.1. However, in one trial, the rat was alsoinjected with 2.5 mg Fe/kg dextran-coated paramagnetic iron oxide(produced following the procedure described in Example 7.1 except thatno divalent salt was used) 15 minutes prior to receiving thesuperparamagnetic material. The T₂ of the subject's blood was measuredperiodically over the subsequent 3 hours. The results are present inFIG. 7.

Briefly, in both trials, the blood T₂ dropped dramatically within 5minutes after the superparamagnetic material was added. However, thevalue rapidly returned to normal in the rat which did not receive thesuperparamagnetic material, presumably due to absorption of the agent bythe reliculoendothelial system (RES). In contrast, when the paramagneticagent was used the T₂ depression is dramatically extended. This is dueto a competition between the superparamagnetic and the paramagneticmaterial for RES receptors, greatly expanding the lifetime of thesuperparamagnetic agent.

It is apparent that many modifications and variations of this inventionas hereinabove set forth may be made without departing from the spiritand scope thereof. The specific embodiments described are given by wayof example only and the invention is limited only by the terms of theappended claims.

What is claimed is:
 1. A method for obtaining an in vivo MR image of anorgan or tissue of an animal or human subject which comprises (a)administering to such animal or human subject an effective amount of acontrast agent in a physiologically acceptable carrier, which contrastagent comprises a biodegradable superparamagnetic metal oxide, saidbiodegradable superparamagnetic metal oxide being characterized bybiodegradation in such subject within about 2 weeks or less afteradministration, as evidenced by a return of the proton relaxation ratesof said organ or tissues to preadministration levels; and (b) obtainingan MR image from such subject.
 2. The method of claim 1 in which saidbiodegradable metal oxide is associated with a polymeric substance. 3.The method of claim 2 in which said polymeric substance is apolysaccharide.
 4. The method of claim 1, 2, or 3 in which said metal isiron.